DNA Sequencing Reveals Three New Species of Chamberlainium

Botanica Marina 2021; 64(1): 19–40

Research article

Courtney A. Puckree-Padua, Paul W. Gabrielson and Gavin W. Maneveldt* DNA sequencing reveals three new species of Chamberlainium (Corallinales, Rhodophyta) from South Africa, all formerly passing under Spongites yendoi https://doi.org/10.1515/bot-2020-0074 are correctly applied; all other non-geniculate coralline Received November 27, 2020; accepted December 18, 2020; names are likely misapplied in South Africa. published online January 6, 2021 Keywords: biogeography; Chamberlainoideae; cryptic Abstract: Three new non-geniculate coralline algal spe- diversity; morpho-anatomy; non-geniculate coralline algae. cies from South Africa are described that were passing under the misapplied name, Spongites yendoi. Based on plastid encoded DNA sequences from psbA and rbcL 1 Introduction markers, these species belong in the subfamily Chamber- lainoideae. The DNA sequences, supported by the morpho- The South African rocky intertidal and shallow subtidal anatomical character of tetrasporangial conceptacle roof zones have both an abundance and diversity of non- development, placed all three species in the genus Cham- geniculate coralline red algae with three (Corallinales, berlainium and not Pneophyllum, the only other genus in Hapalidiales, Sporolithales; Stephenson and Stephenson Chamberlainoideae. In addition to the diagnostic DNA se- 1972; Maneveldt et al. 2008) of the four orders (Corallinales, quences, Chamberlainium capense sp. nov., C. glebosum Corallinapetrales, Hapalidiales, Sporolithales; Jeong et al. sp. nov. and Chamberlainium occidentale sp. nov. may 2020) represented. South Africa has representative species be distinguished by a combination of habit, habitat, from more than half of the currently recognized extant geographic distribution, and several morpho-anatomical genera of non-geniculate coralline red algae, with the order features. Biogeographically all three species are found in Corallinales best represented (Maneveldt et al. 2016). the Benguela Marine Province of South Africa, with Included in this order are species previously placed in C. occidentale being the most widespread. Chamberlainium Spongites and the recently erected Chamberlainium that are glebosum also has a wide, but disjunct distribution particularly widespread and ecologically important in and C. capense is another South African endemic non- South Africa (Chamberlain 1993; Keats et al. 1993; Man- geniculate coralline, whose range is restricted to a 43 km eveldt and Keats 2008; Puckree-Padua et al. 2020b). stretch of coastline. Thus far, DNA sequences from type Based on a multi-gene molecular phylogeny, Rösler specimens of non-geniculate corallines show that only et al. (2016) noted that numerous southern hemisphere those species whose type localities are from South Africa species, assigned to Spongites, did not occur in the same clade with the generitype, Spongites fruticulosus Kützing (type locality: Mediterranean Sea), and would have to be placed in another genus. Caragnano et al. (2018) subse- *Corresponding author: Gavin W. Maneveldt, Department of quently proposed a new genus Chamberlainium and a new Biodiversity and Conservation Biology, University of the Western Cape, P. Bag X17, Bellville 7535, South Africa, subfamily Chamberlainoideae to accommodate sequenced E-mail: [email protected]. https://orcid.org/0000-0002-5656- and named Spongites species from the northeast Paciﬁc 5348 and from South Africa (van der Merwe et al. 2015), making Courtney A. Puckree-Padua, Department of Biodiversity and the new combinations Chamberlainium tumidum (Foslie) Conservation Biology, University of the Western Cape, P. Bag X17, Caragnano, Foetisch, Maneveldt et Payri, Chamberlainium Bellville 7535, South Africa. https://orcid.org/0000-0001-7684-0610 decipiens (Foslie) Caragnano, Foetisch, Maneveldt et Payri Paul W. Gabrielson, Biology Department and Herbarium, University of North Carolina at Chapel Hill, Coker Hall CB 3280, Chapel Hill, NC, and Chamberlainium agulhense (Maneveldt, E.van der 27599-3280, USA. https://orcid.org/0000-0001-9416-1187 Merwe et P.W.Gabrielson) Caragnano, Foetisch, Maneveldt

Open Access. © 2020 Courtney A. Puckree-Padua et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 20 C.A. Puckree-Padua et al.: South African species of Chamberlainium

et Payri. Two morpho-anatomical characters were pro- ecologically important and abundant epilithic intertidal posed to segregate Chamberlainium from Spongites:1) species along the entire South African coast of roughly diameter of tetra/bisporangial conceptacle chambers 3000 km (Chamberlain 1993; Maneveldt and Keats 2008; (<300 µm for Chamberlainium and >300 µm for Spongites); Maneveldt et al. 2008). The species’ morphological vari- and 2) number of cell layers composing roofs of tetra/bis- ability was largely attributed to variable grazing pressure porangial conceptacles (≤8 cell layers for Chamberlainium and/or to sand scouring (Chamberlain 1993; Maneveldt and >8 for Spongites; Caragnano et al. 2018). The former of and Keats 2008). Recently, DNA sequencing has resolved these characters no longer holds true for separating the seven distinct species passing under S. yendoi in South genera morpho-anatomically (Puckree-Padua et al. 2020b). Africa likely none of which are S. yendoi (Puckree-Padua Until recently, three species were recognized in Spon- et al. 2020b). Three of these species have already been gites in South Africa, based on morpho-anatomy, Spongites treated, Chamberlainium agulhense (Caragnano et al. 2018; impar (Foslie) Y.M.Chamberlain (type locality: Cape van der Merwe et al. 2015), Chamberlainium cochleare Peninsula, Western Cape Province, South Africa), Spongites Puckree-Padua, Haywood, P.W.Gabrielson et Maneveldt, discoideus (Foslie) Penrose et Woelkerling (type locality and Chamberlainium natalense (Foslie) Puckree-Padua, mouth of the Rio Grande, Tierra del Fuego, Argentina), and P.W.Gabrielson et Maneveldt (Puckree-Padua et al. 2020b). Spongites yendoi (Foslie) Y.M.Chamberlain (type locality: Herein we describe, name, and transfer to Chamberlainium Shimoda Harbor, Shimoda Prefecture, Japan). Spon- three additional species. The seventh species, geographi- gites impar was transferred to Chamberlainium as Cham- cally distinct, which also was passing under S. yendoi, will berlainium impar (Foslie) Puckree-Padua, P.W.Gabrielson be transferred later. et Maneveldt (Puckree-Padua et al. 2020b). The lectotype of S. discoideus was sequenced and shown to be distinct from South African specimens given that name and to belong in 2 Materials and methods Pneophyllum to which it was transferred as Pneophyllum discoideum (Foslie) Puckree-Padua, Maneveldt et Specimens were collected during spring low tides from the rocky P.W.Gabrielson (Puckree-Padua et al. 2020a). An existing intertidal and shallow subtidal at various locations along the South name was available for the South African S. discoideus African coastline (Figure 1). Additional specimens from Namibia (southern West Africa), housed in the UWC herbarium, were also specimens, Lithophyllum marlothii (Heydrich) Heydrich, examined. Specimens on bedrock were removed using a geological whose type specimen was also sequenced, and it too was hammer and chisel. Where specimens occurred on pebbles or transferred to Pneophyllum as Pneophyllum marlothii gastropod shells, the entire pebble or shell along with the coralline (Heydrich) Puckree-Padua, P.W.Gabrielson, Hughey et was sampled. Specimens were sorted in the ﬁeld. For each specimen, ﬁ Maneveldt (Puckree-Padua et al. 2020a). one fragment was air-dried, one placed in silica gel, and one xed in 10% formalin. In South Africa, based on morpho-anatomy, S. yendoi Specimens for sequencing were examined and prepared was considered to be variable in morphology (thin and following Gabrielson et al. (2011) and extracted following Hughey et al. featureless to thick and protuberant) and the most (2001). Voucher specimens were deposited in UWC and NCU. Type

Figure 1: Map of South Africa (gray shade), with an inset of False Bay, showing the coastal provinces, the biogeographic marine provinces and the approximate locations of collecting sites (● = Chamberlainium capense, ■ = C. glebosum, : = C. occidentale). C.A. Puckree-Padua et al.: South African species of Chamberlainium 21

specimens were deposited in L (Naturalis, Netherlands, Leiden). and 24 from GenBank) were used in the phylogenetic an- Herbarium acronyms followed Thiers (2020, continuously updated). alyses (Supplementary Table S1). The RAxML and Bayesian Specimens for light microscopy were prepared following Man- analyses, for both the psbA (Figure 2) and rbcL (Figure 3) eveldt and van der Merwe (2012). In cell measurements, length denotes the distance between primary pit connections, and diameter the genes were congruent in resolving Chamberlainium maximum width of the cell lumen at right angles to this. Conceptacle capense sp. nov., Chamberlainium glebosum sp. nov. and measurements followed Adey and Adey (1973). The determination and Chamberlainium occidentale sp. nov. in a monophyletic presentation (i.e., ranges and not averages with standard deviations/ clade in Chamberlainoideae with full support (100% BS/ errors) of all measurements followed Maneveldt et al. (2017). Thallus 1 PP). These three species occurred in a clade with the anatomical terminology followed Chamberlain (1990). Growth form generitype of Chamberlainium, C. tumidum, in both psbA terminology, e.g., encrusting, warty, lumpy, followed Woelkerling et al. (1993). (82/0.97, Figure 2) and rbcL (99/1, Figure 3) trees. In the Amplification of the rbcL marker was according to Gabrielson psbA tree, C. glebosum and Chamberlainium capsense are et al. (2011) and of the psbA marker according to Sissini et al. (2014). sister species with moderate support (76/0.93) with Resulting PCR products were sent to the DNA Analysis Core Facility at C. occidentale sister to that pair with full support, whereas the University of North Carolina at Wilmington for sequencing and in the rbcL tree, C. occidentale and C. capense are sister were manually aligned and compiled using Sequencher (Gene Codes Corp., Ann Arbor, Michigan, USA). Sequenced specimens are listed in species with strong support (96/1) with C. glebosum sister to Supplementary Table S1. that pair again with full support. Both genes show support Used for the phylogenetic analyses were: 11 (of 31 – identical sequences removed) psbA sequences and 15 (of 24 – identical sequences removed) rbcL sequences from specimens previously assigned to S. yendoi from South Africa; six psbA and rbcL sequences from unknown species within Chamberlainoideae from Chile and Antarctica; one psbA sequence from a specimen assigned to Pneo- phyllum fragile Kützing from New Zealand; seven psbA and rbcL sequences from established species of Chamberlainium; three psbA and one rbcL sequence from established species of Pneophyllum; and an S. fruticulosus psbA sequence (Supplementary Table S1). The rbcL and psbA sequences from the orders Gelidiales (one), Sporolithales (three), Hapalidiales (four and three respectively) and Corallinales (two) were used as outgroups in order to provide phylogenetic placement of Chamberlainoideae. Sequences from P. fragile specimens, from Chamberlainium specimens (van der Merwe et al. 2015) and outgroup sequences were obtained from GenBank. Outgroup sequence lengths ranged from 293 to 1364 bp. The remaining psbA and rbcL sequences ranged between 629–851 bp and 691–1387 bp long, respectively. Se- quences for each gene were aligned using MUSCLE (Edgar 2004), as implemented in Geneious, with a maximum of eight iterations. Geneious v11.1.5 (https://www.geneious.com, Kearse et al. 2012) was used for all phylogenetic analyses. Identical sequences were removed from the respective datasets before Maximum Likelihood (RAxML, Stamatakis 2006) analyses were performed on 38 psbA and 39 rbcL sequences separately using the GTR CAT model of evolution applying the Rapid-hill climbing algorithm with 10 topological searches from random starts and then Rapid Bootstrapping and search for best-scoring ML tree algorithm, with 500 bootstrap replicates. Figure 2: Maximum likelihood tree based on psbA sequences. Bayesian analyses were performed on 38 unique psbA and 39 Species with sequenced type/‘topotype’ material are highlighted in unique rbcL sequences using the Geneious v11.1.5 (https://www. bold. Generitype species and molecular references for generitype geneious.com, Kearse et al. 2012) MrBayes v3.2.6 plugin (Huelsenbeck species within the Corallinales are identiﬁed by a star (+). Species and Ronquist 2001). Under the GTR model, sampling was performed names are followed by their GenBank accession number and their every 1000 generations. Analyses were run for 1.1 million generations geographic location, including ocean basin where needed. Numbers with a 100 000 burn-in. in brackets, following the GenBank accession numbers, are the total number of identical sequences. Bootstrap support (BS) and Bayesian posterior probability (BPP) values are provided at branch 3 Results nodes. BS and BPP values = 100% and = 1 respectively, are denoted by an asterisk (*); BS and BPP values <75 % and < 0.75 respectively, are not shown. Atl = Atlantic Ocean, Ind = Indian Ocean, A total of 58 psbA sequences (31 newly generated and 27 Med = Mediterranean, Nam = Namibia, NZ = New Zealand, from GenBank) and 48 rbcL sequences (24 newly generated Pac = Paciﬁc Ocean. 22 C.A. Puckree-Padua et al.: South African species of Chamberlainium

Figure 3: Bayesian inference tree based on rbcL sequences. Species with sequenced type/‘topotype’ material are highlighted in bold. Generitype species within Corallinales are identified by a star (+). Species names are followed by their GenBank accession number and their geographic location, including ocean basin where needed. Numbers in brackets, following the GenBank accession numbers, are the total number of identical sequences. Bootstrap support (BS) and Bayesian posterior probability (BPP) values are provided at branch nodes. BS and BPP values = 100 % and = 1 respectively, are denoted by an asterisk (*); BS and BPP values < 75 % and < 0.75 respectively. Atl = Atlantic Ocean, Ind = Indian Ocean, Med = Mediterranean, Pac = Pacific Ocean. for C. cochleare sister to the C. capense + C. glebo- Chamberlainium capense Puckree-Padua, P.W. sum + C. occidentale clade, moderate for psbA (83/0.79), Gabrielson et Maneveldt sp. nov. (Figures 4, 7–11, 12–14, full for rbcL (100/1). In the psbA tree (Figure 2), the clade 15–18; Tables 1, 2). formed by C. cochleare, C. capense, C. glebosum and Holotype: L 3986119, 09.x.2016, leg. G.W. Maneveldt, C. occidentale is weakly supported (83/0.79) as sister to two collection number 16/05, epilithic on primary bedrock in northeast Pacific species, C. tumidum and C. decipiens;in mid-intertidal, sand inundated rock pool. the rbcL tree (Figure 3) the relationship of these northeast Type locality: South Africa, Western Cape Province, Pacific species is unresolved with respect to the other Mouille Point (33°53.9440′ S, 18°24.5285′ E). Chamberlainium species. Etymology: ‘capense’ in reference to the species’ Interspecific sequence divergences for the psbA gene restricted distribution along the Cape Peninsula histori- ranged from 1.71 to 11.59% for Chamberlainium species, cally known as the ‘Cape of Good Hope, South Africa’ whereas intraspecific sequence divergences ranged from (Caput Bonae Spei). 0 to 1.41% (Supplementary Table S2). For psbA, C. capense Description: Non-geniculate, thalli are moderately differed from C. glebosum by 3.14%, from C. occidentale by thick (up to 1000 µm), encrusting to mostly variably lumpy 2.84%, and C. glebosum from C. occidentale by 3.51%. to slightly protuberant. Thalli are epilithic or epizoic and Interspecific sequence divergences for the rbcL gene bright to dusky pink in well-lit conditions. Individual crusts ranged from 0.98 to 12.19% for Chamberlainium species, do not appear to fuse together and are easily discernible. whereas intraspecific sequence divergences ranged from Thallus construction is monomerous with a single layer of 0 to 0.17% (Supplementary Table S3). For rbcL, C. capense epithallial cells. A central columella is present in tetra- differed from C. glebosum by 2.25%, from C. occidentale by sporangial conceptacles that disintegrate to form a low 0.98%, and C. glebosum from C. occidentale by 2.68%. mound with maturity. The pore opening in mature tetra- For all taxa, morpho-anatomy was done only on sporangial conceptacles is occluded by a corona of fila- sequenced specimens. See Supplementary Table S4 for ments that projects above the opening. The psbA (851 bp) collection details. and rbcL (691–1387 bp) sequences are diagnostic. C.A. Puckree-Padua et al.: South African species of Chamberlainium 23

Figures 7–11: Chamberlainium capense habit and vegetative anatomy. (7) Rock fragment showing holotype specimen (white arrow) (L 3986119, tetrasporangial). Scale bar = 20 mm. (8) Encrusting to variably lumpy and slightly protuberant, epilithic thalli showing crusts abutting and easily discernible. Scale bar = 10 mm. (9) Vertical section through the margin (black arrow) showing the Figures 4–6: Habit photographs of Chamberlainium capense, monomerous thallus construction with plumose medulla (M) giving C. glebosum and C. occidentale. (4) Chamberlainium capense is rise to cortical filaments (C) that terminate in a single layer of moderately thick to mostly lumpy and only slightly protuberant. epithallial cells (black arrowhead) (UWC 16/17). Scale bar = 50 μm. Scale bar = 20 mm. (5) Chamberlainium glebosum is thick, very (10) Vertical section of the inner thallus showing cell fusions lumpy and highly protuberant. Scale bar = 20 mm. (6) (f) between adjacent medullary filaments (UWC 16/17). Scale Chamberlainium occidentale is morphologically highly variable, bar = 20 μm. (11) Vertical section of the outer thallus showing a occurring as thin encrusting (smooth) to thick warty (mostly) to single layer of epithallial cells (e) subtended by a layer of lumpy plants, and are only slightly protuberant. Scale bar = 30 mm. subepithallial initials (i). Note the paired, bottle-shaped trichocytes (t) (UWC 16/24). Scale bar = 20 μm. Habitat: Thalli were epilithic on the primary bedrock and on large boulders in rock pools and on exposed plat- Thalli were dorsiventrally organized, monomerous forms in the mid-intertidal zone, as well as epizoic on and haustoria were absent. The medulla was thin and mollusc shells in the low intertidal zone. plumose (non-coaxial) (Figures 9, 10). Medullary filaments Vegetative morphology and anatomy: Thalli were comprised rectangular to elongate cells, which gave rise to non-geniculate, moderately thick (up to 1000 μm), cortical filaments that comprised mainly square to rect- encrusting (smooth) to mostly variably lumpy and slightly angular cells (Figure 10). Contiguous medullary and protuberant, with protuberances to 4 mm tall (Figures 4 cortical filaments were joined by cell fusions; secondary pit and 7, 8), and were firmly adherent, dusky pink to mauve connections were absent (Figure 10). Subepithallial initials (in well-lit conditions) to rosy or purple-pink (in dim light) (intercalary meristematic cells) were square to rectangular when freshly collected (Figure 4). Individual crusts did not (Figure 11). The epithallus was single layered with oval to fuse together and were easily discernible (Figures 7, 8). rounded cells (Figure 11). Trichocytes were common at the 24 C.A. Puckree-Padua et al.: South African species of Chamberlainium

(Figure 12). This protective layer was shed once the pore canal was near fully developed. The pore opening was occluded by a mucilage plug (Figures 13, 14). In mature conceptacles, terminal initials along pore canal were enlarged and papillate; they projected into the pore canal and were orientated more or less parallel to the conceptacle roof surface (Figure 14). Unbranched (simple) spermatangial systems were confined to mature conceptacle floor (Figures 12–14). Senescent male conceptacles appeared to be shed; no buried conceptacles were observed. Tetrasporangial thalli were morphologically similar to spermatangial thalli. Conceptacles were uniporate, low domed and raised above the surrounding thallus surface (Figures 15–17). Conceptacle chambers were transversely elliptical to bean-shaped. Conceptacle roof was nearly twice as thick along the pore canal and was 5–7 cells thick, including an epithallial cell. Pore canal tapered towards the surface, formed an arch (Figure 18) and was lined by elongated papillate cells that projected into the canal and Figures 12–14: Chamberlainium capense spermatangial anatomy were orientated more or less parallel or nearly perpendic- (UWC 16/21). (12) Vertical section through the outer thallus showing ular to the conceptacle roof surface (Figure 18). The roof a spermatangial conceptacle primordium with peripheral roof was formed from filaments peripheral to the fertile area development (black arrows) and simple, spermatangial systems (Figure 15) and terminal initials were more elongate than (black arrowheads) confined to the conceptacle floor. Note the protective layer of epithallial cells (white arrow). Scale bar = 50 μm. their inward derivatives. Throughout early development a (13) Vertical section through a mature, raised spermatangial protective layer of epithallial cells surrounded the conceptacle showing the mucilage plug (white arrowhead) that conceptacle primordium (Figure 15). This protective layer occludes the pore opening and simple, spermatangial systems was shed once pore canal was near fully developed; the (black arrowheads) confined to the conceptacle floor. Scale pore opening was occluded by a corona of filaments that bar = 50 μm. (14) Magnified view through a mature spermatangial conceptacle showing the mucilage plug (white arrowhead) that projected above the pore opening (Figures 16, 18). The occludes the pore opening and simple, spermatangial systems corona appeared to form from filaments near the upper half (black arrowheads) confined to the conceptacle floor. Note that the of the roof, directly adjacent to the pore canal (Figures 16, pore canal is lined with terminal, elongate initials (black arrows) that 18). Throughout the development of the tetrasporangial μ project into the pore canal as papillae. Scale bar = 20 m. conceptacle, a prominent columella of sterile filaments formed at the center of the conceptacle chamber thallus surface and occurred singularly (mostly) to paired (Figure 15), which extended into the pore canal (Figure 16); (Figure 11). Trichocytes were always terminal and never the central columella appeared weakly calcified as with intercalary in the cortex; buried trichocytes were not maturity it disintegrated to form a low mound (Figure 17). observed. Data on morphological and measured vegetative The base of the pore canal was sunken into the chamber characters are summarized in Table 1. and terminal initials near the base pointed downward Reproductive morphology and anatomy: Game- (Figure 18). Mature conceptacle floors were sunken 11– tangial thalli appeared to be dioecious, although female 17 cells (including the epithallial cell) below the sur- plants were not observed. rounding thallus surface. Zonately divided tetrasporangia Spermatangial (male) conceptacles were uniporate, were arranged at the extreme periphery of the conceptacle low-domed and raised above surrounding thallus surface chamber and were attached via a stalk cell (Figures 16, 17). (Figures 12–14). Conceptacle chambers were transversely Where the central columella had disintegrated or dimin- elliptical to flatten; conceptacle roof nearly twice as thick ished, the tetrasporangia filled the chamber and appeared along pore canal (Figures 13, 14). Conceptacle roof formed to be distributed across the chamber floor (Figure 17). Se- from filaments peripheral to the fertile area (Figure 12). nescent tetrasporangial conceptacles appeare to be shed as Throughout the early development, a protective layer of no buried conceptacles were observed. Data on reproduc- epithallial cells surrounded the conceptacle primordium tive characters are summarized in Table 2. C.A. Puckree-Padua et al.: South African species of Chamberlainium 25

Figures 15–18: Chamberlainium capense tetrasporangial anatomy. (15) Vertical section through the outer thallus showing a later stage tetrasporangial conceptacle primordium with peripheral roof development (black arrowheads) and tetrasporangial initials (t) arranged peripherally around a central columella (c). Note the persisting layer of protective epithallial cells (black arrow) (L 3986119). Scale bar = 50 μm. (16) Vertical section through a mature, raised tetrasporangial conceptacle showing tetrasporangia (t) with a stalk cell (black arrowhead), peripherally arranged around a well-defined central columella (C). Note the remains of a corona (white arrow) that surrounds the pore opening (L 3986119). Scale bar = 50 μm. (17) Vertical section through a mature raised tetrasporangial conceptacle showing tetrasporangia (t) with stalk cells (black arrowheads), appearing to be arranged across the chamber floor as the columella (c) has disintegrated. Note the absence of the corona (white arrow) (UWC 16/09). Scale bar = 50 μm. (18) Magnified view of the pore canal of a mature tetrasporangial conceptacle showing the base of the pore canal sunken (black arrowheads) into the chamber with terminal, elongate initials near the base pointing downward (black arrows) and the remains of a corona (white arrow) that surrounds and occludes the pore opening. (L 3986119). Scale bar = 20 μm.

Distribution: Conﬁrmed by DNA sequences to have a Description: Non-geniculate, thalli are thick (to restricted distribution (±43 km distance) along the 2000 µm), very lumpy becoming highly protuberant. Thalli southwest coast, from Mouille Point to Slangkop (West- are epilithic and brownish pink to gray in well-lit condi- ern Cape Province) along the Cape Peninsula, South tions. Individual crusts do not appear to coalesce (do not Africa. fuse together) and are easily discernible. The thallus con- Chamberlainium glebosum Puckree-Padua, P.W. struction is monomerous with a single layer of epithallial Gabrielson et Maneveldt sp. nov. (Figures 5, 19–23, cells. A central columella is present in tetrasporangial 24–25, 26–30; Tables 1, 2). conceptacles, which disintegrates to form a low mound Holotype: L 3986123, 17vii.2015, leg. G.W. Maneveldt, with maturity. The pore opening in mature tetrasporangial collection number 18/05, epilithic on primary bedrock in a conceptacles is unoccluded. The psbA (851 bp) and rbcL mid-intertidal rock pool. (691–1387 bp) gene sequences are diagnostic. Type locality: South Africa, Western Cape Province, Habitat: Thalli were epilithic on the primary bedrock Melkbosstrand (33°44.2518′ S, 18°26.1343′ E). in the high and mid-intertidal zones and only occasionally Etymology: ‘glebosum’ from ‘glebosus’, making refer- on the low shore. ence to the species’ lumpy (Stearn 1973) and highly pro- Vegetative morphology and anatomy: Thalli were tuberant growth form. non-geniculate, thick (up to 2000 μm), very lumpy Table : Comparison of the appearance and vegetative structure of South African species formally ascribed to Chamberlainium. 26

Character Chamberlainium Chamberlainium Chamberlainium cochle- Chamberlainium Chamberlainium Chamberlainium nata- Chamberlainium occi- agulhense (van der capemse (this study) are (Puckree-Padua et al. glebosum (this impar (Puckree- lense (Puckree-Padua dentale (this study) ..PcrePdae l:SuhArcnseisof species African South al.: et Puckree-Padua C.A. Merwe et al. ) b) study) Padua et al. b) et al. b)

Growth form Encrusting (smooth) Encrusting (smooth), Encrusting (smooth) Lumpy, becoming Encrusting (smooth), Encrusting (smooth) Encrusting (smooth) to becoming variably highly protuberant orbicular becoming warty (mostly) to lumpy, lumpy (mostly) and conﬂuent becoming slightly slightly protuberant protuberant Thallus Dimerous Monomerous Monomerous Monomerous Monomerous Monomerous Monomerous construction Maximum thallus        thickness Habit Individual thalli Individual thalli Individual thalli not Individual thalli Individual thalli Individual thalli discern- Individual thalli discern- discernible, not discernible, not fusing discernible, fusing to discernible, not discernible, not ible, not fusing ible, not fusing fusing form large expanses fusing fusing Trichocytes None observed Common, solitary Common, solitary None observed Rare, solitary to Rare, solitary (mostly) to Common, solitary (mostly) to paired (mostly) to clustered, up paired clustered, up to  (sepa- (mostly) to clustered, up to  (separated by normal rated by normal vegeta- to  (separated by normal

vegetative filaments) tive filaments) vegetative filaments) Chamberlainium Trichocyte - L = –; D = – L = –; D = – - L = –; D = – L = –; D = – L = –; D = – dimensions  No. of epithallial – (mostly –)  – ()  cell layers Epithallial cell Rounded to domed Oval to round Elliptical to domed Rounded to Elliptical to rounded Flattened to elliptical Elliptical to rounded to shape elliptical elongate Epithallial cell ND L = –; D = – L = –; D = – L = –; D = – L = –; D = – L = –; D = – L = –; D = – dimensions Subepithallial cell Square to rectangular Square to rectangular Square to rectangular Rectangular Square to Square to rectangular Rectangular shape rectangular Subepithallial cell ND L = –; D = – L = –; D = – L = –; D = – L = –; D = – L = –; D = – L = –; D = – dimensions Cortical cell shape - Square to rectangular Square to rectangular Square to Square to beaded Square to rectangular Square to rectangular rectangular Cortical cell - L = –; D = – L = –; D = – L = –; D = – L = –; D = – L = –; D = – L = –; D = – dimensions Medullary cell - Rectangular Rectangular to elongate Rectangular to Rectangular to Rectangular to elongate Square to rectangular shape elongate elongate Medullary cell - L = –; D = – L = –; D = – L = –; D = – L = –; D = – L = – D = – L = –; D = – dimensions  Erect filament cell Square to rectangular ------shape with rounded corners Table : (continued)

Character Chamberlainium Chamberlainium Chamberlainium cochle- Chamberlainium Chamberlainium Chamberlainium nata- Chamberlainium occi- agulhense (van der capemse (this study) are (Puckree-Padua et al. glebosum (this impar (Puckree- lense (Puckree-Padua dentale (this study) Merwe et al. ) b) study) Padua et al. b) et al. b)

Erect filament cell ND ------dimensions Basal filament cell Non-palisade, irregu------shape (in radial larly square view) Basal filament cell L = –; H = – ------dimensions (in radial view) Basal filament cell Rectangular to pali------shape (in tangen- sade-like tial view) Basal filament cell L = –; H = – ------dimensions (in tangential view) of species African South al.: et Puckree-Padua C.A.

Unless otherwise stated, all measurements are in micrometers. L, H and D, length, height and diameter, respectively; ND, no data provided. Chamberlainium 27 Table : Comparison of the reproductive structures of South African species formally ascribed to Chamberlainium. 28

Character Chamberlainium Chamberlainium Chamberlainium coch- Chamberlainium Chamberlainium Chamberlainium nata- Chamberlainium occi- (van der (this study) (Puckree-Padua (this (Puckree-Padua (Puckree-Padua (this study) agulhense capense leare glebosum impar lense dentale of species African South al.: et Puckree-Padua C.A. Merwe et al. ) et al. b) study) et al. b) et al. b)

Mature tetraspor- Pore opening unoc- Pore opening occluded Pore opening occluded Pore opening unoc- Pore opening Pore opening occluded Pore opening unoc- angial pore cluded and flush by a corona of fila- by a mucilage plug and cluded and flush occluded by a muci- by a corona of filaments cluded and slightly characteristics with surrounding ments and flush with flush with surrounding with surrounding lage plug and flush and flush with sur- sunken below the sur- roof surrounding roof roof roof with surrounding roof rounding roof rounding roof Thickness of mature – – – – – – – tetrasporangial conceptacle roof No. of cells (incl. epi- ––––––– thallial cells) in mature tetrasporangial conceptacle roof Depth (no. of cells) of – – – – – – – mature tetrasporangial conceptacle Chamberlainium floor Shape of pore canal Pore canal not arch- Pore canal arching, Pore canal not arching, Pore canal not arch- Pore canal not arch- Pore canal not arching, Pore canal arching, ing, tapered towards tapered towards tapered towards the ing, tapered towards ing, tapered towards tapered towards the tapered towards surface surface surface or straight in surface surface surface or straight in surface senescing very mature conceptacles conceptacles Tetrasporangia ND L = –; D = – L = –; D = – L = –; D = – L = –; D = – L = –; D = – L = –; D = – dimensions  Tetrasporangia Peripheral Peripheral, may appear Peripheral Peripheral Peripheral Peripheral, may appear Peripheral, may appear arrangement distributed across distributed across distributed across chamber floor as colu- chamber floor as colu- chamber floor as columella disintegrates mella disintegrates mella disintegrates Central columella Prominent; disinte- Prominent; disinte- Prominent; disinte- Prominent; disinte- Prominent; persists to Prominent; disinte- Prominent; disintegrates to form a low grates to form a low grates to form a low grates to form a low maturity grates to form a low grates to form a low mound with maturity mound with maturity mound with maturity mound with maturity mound with maturity mound with maturity Conceptacles buried Shed Shed Shed Shed Shed Conceptacles mostly Shed or shed shed; occasionally tetrasporangia and sper- matangia may be found buried

Unless otherwise stated, all measurements are in micrometers. L and D, length and diameter respectively; ND, no data provided. C.A. Puckree-Padua et al.: South African species of Chamberlainium 29

connections were absent (Figures 22, 23). Subepithallial initials (intercalary meristematic cells) were square to rectangular (Figure 23). The epithallus was single layered with rounded to elliptical cells (Figure 23). Trichocytes were not observed. Data on morphological and measured vegetative characters are summarized in Table 1. Reproductive morphology and anatomy: Game- tangial thalli appeared to be dioecious, although female plants were not observed. Spermatangial (male) conceptacles were uniporate, low-domed, raised above surrounding thallus surface (Figures 24, 25). Conceptacle chambers were transversely elliptical to flatten with the roof nearly twice as thick along the pore canal (Figure 25). The roof was formed from filaments peripheral to the fertile area (Figure 24). Throughout the early development, a protective layer of epithallial cells surrounded the conceptacle primordium (Figure 24). This protective layer was shed once the pore canal was near fully developed. The pore opening was occluded by a mucilage plug (Figure 25). In mature conceptacles the terminal initials along the pore canal were enlarged and papillate, they projected into the pore canal and were orientated more or less parallel to the conceptacle roof surface (Figure 25). Unbranched (simple) spermatangial Figures 19–23: Chamberlainium glebosum habit and vegetative systems were confined to floor of mature conceptacle anatomy. (19) Rock fragment with holotype specimen showing the (Figures 24, 25). Senescent male conceptacles appeared to region (white arrow) of the holotype from which all analyses were be shed as no buried conceptacles were observed. done (L 3986123, tetrasporangial). Scale bar = 20 mm. (20) Magnified view showing highly protuberant nature of C. glebosum Tetrasporangial thalli were morphologically similar to holotype specimen (L 3986123). Scale bar = 2 mm. (21) Vertical spermatangial thalli. Conceptacles were uniporate, low section through the margin (black arrow) showing the monomerous domed and raised above surrounding the thallus surface thallus construction with plumose medulla (M) giving rise to cortical (Figures 26–29). Conceptacle chambers were transversely fi laments (C) that terminate in a single layer of epithallial cells (black elliptical to bean-shaped. The roof was nearly twice as arrowhead) (UWC 15/34). Scale bar = 100 μm. (22) Vertical section of thick along the pore canal and were 4–8 (9) cells (including the inner thallus showing cell fusions (f) between adjacent medullary filaments (UWC 15/34). Scale bar = 10 μm. (23) Vertical an epithallial cell) thick (Figure 29). The pore canal tapered section of the outer thallus showing a single layer of epithallial cells towards the surface was lined by elongated papillate cells (e) subtended by a layer of subepithallial initials (i). Note the cell that projected into the pore canal and were orientated more fusions (f) between adjacent cortical filaments (UWC 15/34). Scale or less parallel or nearly perpendicular to the conceptacle μ bar = 20 m. roof surface (Figure 30). The roof was formed from filaments peripheral to the fertile area (Figures 26–28) and becoming highly protuberant with protuberances to 6 mm terminal initials were more elongate than their inward tall (Figures 5, 19 and 20), and were firmly adherent, mostly derivatives (Figure 27). Throughout the early development brownish pink to greyish when freshly collected (Figure 5). a protective layer of epithallial cells surrounded the Individual crusts did not coalesce (did not fuse together) conceptacle primordium (Figures 26–28). This protective and were easily discernible (Figure 5). layer was shed once the pore canal was near fully devel- Thalli were dorsiventrally organized, monomerous oped. The pore opening in mature conceptacles was and haustoria were absent. The medulla was thin and unoccluded (Figures 29, 30). Throughout the development plumose (non-coaxial) (Figures 21, 22). Medullary filaments of the immature tetrasporangial conceptacle, a prominent comprised rectangular to elongate cells, which gave rise to columella of sterile filaments formed at the center of the cortical filaments that comprised square to rectangular conceptacle chamber (Figures 27, 28), which extended into cells (Figures 22, 23). Contiguous medullary and cortical the pore canal (Figure 29); the central columella appeared filaments were joined by cell fusions; secondary pit weakly calcified as with maturity it disintegrated to form a 30 C.A. Puckree-Padua et al.: South African species of Chamberlainium

Figures 24–25: Vertical section through the outer thallus showing spermatangial conceptacle primordium with peripheral roof development (black arrows) and simple spermatangial systems (black arrowheads) confined to the conceptacle floor. Note the protective layer of epithallial cells (white arrow). Scale bar = 50 μm. Vertical section through the outer thallus showing a mature, raised spermatangial conceptacle with the mucilage plug (white arrow) that occludes the pore opening and simple, spermatangial systems (white arrowheads) confined to the conceptacle floor. Note that the pore canal is lined with terminal, elongate initials (black arrowheads) that project into the pore canal as papillae. Scale bar = 50 μm. low mound. The base of the pore canal was sunken into the not fuse together) and are easily discernible. The thallus chamber and terminal initials near the base pointed construction is monomerous with a single layer of epi- downward (Figures 29, 30). Mature conceptacle floors were thallial cells. A central columella is present in tetraspor- sunken 11–24 cells (including the epithallial cell) below the angial conceptacles, which persists to maturity. The pore surrounding thallus surface. Zonately divided tetrasporangia opening in mature tetrasporangial conceptacles is unoc- at maturity were arranged at the extreme periphery of cluded and slightly sunken below the surrounding roof conceptacle chamber and was attached via a stalk cell surface. The psbA(851bp)andrbcL (691–1,384 bp) (Figure 29). Senescent tetrasporangial conceptacles appeared sequences are diagnostic. to be shed as no buried conceptacles were observed. Data on Habitat: Thalli were epilithic on the primary bedrock reproductive characters are summarized in Table 2. in the high, mid- and low intertidal zones and epizoic on Distribution: Confirmed by DNA sequences to have a mollusc shells in the low intertidal zone. wide, but disjunct distribution along the west coast, Vegetative morphology and anatomy: Plants were occurring from Port Nolloth to Groenriviermond (Northern non-geniculate, thalli were variably thin to thick (up to Cape Province, ±220 km distance) and then from Cape St. 1000 μm), encrusting (smooth) to warty (mostly) to lumpy, Martin to Slangkop (Western Cape Province, ±200 km becoming only slightly protuberant (protuberances 4 mm distance), South Africa. in height) (Figures 6, 31). Thalli were firmly adherent, Chamberlainium occidentale Puckree-Padua, P.W. brownish beige (in well-lit conditions) (Figures 6) to blue- Gabrielson et Maneveldt sp. nov. (Figures 6, 31–35, gray to rosy pink (in dim light) when freshly collected. 36–41, 42–46; Tables 1, 2). Individual crusts did not coalesce (did not fuse together) Holotype: L 3986120, 01.x.2015, leg. G.W. Maneveldt and were easily discernible (Figure 31). and C.A. Puckree-Padua, collection number 15/61B, epi- Thalli were dorsiventrally organized, monomerous lithic on primary bedrock in low intertidal zone. and haustoria were absent. The medulla was thin and Isotypes: UWC 15/58, UWC 15/59, UWC 15/61A plumose (non-coaxial) (Figures 32, 33). Medullary fila- Type locality: South Africa, Western Cape Province, ments comprised square to elongate cells, which gave rise Lamberts Bay (32°6.5728′ S, 18°18.1270′ E). to cortical filaments that comprised square to rectangular Etymology: ‘occidentale’ from ‘occidentalis’, meaning cells (Figures 33, 34). Contiguous medullary and cortical western (Stearn 1973), making reference to the species’ wide- filaments were joined by cell fusions; secondary pit con- spread distribution along the west coast of Southern Africa. nections were absent (Figures 33, 34). Subepithallial ini- Description: Non-geniculate, thalli are variably thin tials were square to rectangular (Figure 35). The epithallus to thick (up to 1000 µm), encrusting (smooth) to warty was single layered with elliptical to round to elongate cells (mostly) to lumpy, becoming only slightly protuberant. (Figure 35). Trichocytes were common, mostly singularly, Thalli are epilithic to epizoic and brownish beige in well-lit but also in clusters of up to 6 cells, separated by normal conditions. Individual crusts do not appear to coalesce (do vegetative filaments (Figure 35). Trichocytes were always C.A. Puckree-Padua et al.: South African species of Chamberlainium 31

Figures 26–30: Chamberlainium glebosum tetrasporangial anatomy. (26) Vertical section through the outer thallus showing an early stage tetrasporangial conceptacle primordium with peripherally arranged tetrasporangial initials (black arrowheads) and a protective layer of epithallial cells (black arrow) (UWC 15/17). Scale bar = 50 μm. (27) Vertical section through the outer thallus showing a later stage tetrasporangial conceptacle primordium with peripheral roof development (black arrowheads) with a developing central columella (white arrowhead). Note the persisting layer of protective epithallial cells (black arrow) (UWC 15/17). Scale bar = 50 μm. (28) Vertical section through a maturing tetrasporangial conceptacle showing tetrasporangial initials (t) arranged peripherally around a central columella (between white arrowheads). Note the layer of protective epithallial cells (black arrow) being shed (L 3986123). Scale bar = 50 μm. (29) Vertical section through a mature, raised tetrasporangial conceptacle showing tetrasporangia (t) with stalk cells (white arrowheads), peripherally arranged around a well-defined central columella (C). Note the base of the pore canal sunken into the chamber with terminal, elongate initials near the base pointing downward (black arrowheads) (UWC 15/55). Scale bar = 100 μm. (30) Magnified view of the pore canal of a mature tetrasporangial conceptacle showing terminal, elongate initials (black arrows) that project into the pore canal as papillae and the base of the pore canal sunken (black arrowheads) into the chamber with terminal, elongate initials near the base pointing downward (UWC 15/55). Scale bar = 20 μm. terminal and never intercalary in the cortex; buried tri- canal was near fully developed. The pore opening was chocytes were not observed. Data on morphological and occluded by a mucilage plug (Figure 37). In mature con- measured vegetative characters are summarized in Table 1. ceptacles terminal initials along the pore canal were Reproductive morphology and anatomy: Game- enlarged and papillate, they projected into the pore canal tangial thalli were dioecious and monoecious. The type and were orientated more or less parallel to the conceptacle specimen bore all (including tetrasporangial) life cycle roof surface (Figure 38). Unbranched (simple) sperma- stages. tangial systems were confined to the floor of the mature Spermatangial (male) conceptacles were uniporate, conceptacle (Figures 36, 37). Senescent male conceptacles low-domed, and raised above surrounding thallus surface appeared to be shed as no buried conceptacles were (Figures 36, 37). Conceptacle chambers were transversely observed. elliptical to flatten and the roof nearly twice as thick along Carpogonial (female) conceptacles were similar in size the pore canal (Figures 37, 38). The roof was formed from and shape to spermatangial conceptacles and were raised filaments peripheral to the fertile area (Figure 36). above surrounding thallus surface (Figure 39). In mature Throughout the early development, a protective layer of conceptacles the terminal initials along the pore canal epithallial cells surrounded the conceptacle primordium were enlarged and papillate, they projected into the pore (Figure 36). This protective layer was shed once the pore canal and were orientated more or less parallel or nearly 32 C.A. Puckree-Padua et al.: South African species of Chamberlainium

Figures 31–35: Chamberlainium occidentale habit and vegetative anatomy. (31) Rock fragment with holotype specimen showing the Figures 36–41: Chamberlainium occidentale gametangial anatomy. region (white arrow) of the holotype from which all analyses were (36) Vertical section through the outer thallus showing a spermatangial done (L 3986120, gametangial and tetrasporangial). Scale conceptacle primordium with peripheral roof development (black bar = 10 mm. (32) Vertical section through the margin (black arrow) arrows) and simple spermatangial systems (black arrowheads) showing the monomerous thallus construction with plumose confined to the conceptacle floor. Note the protective layer of medulla (M) giving rise to cortical filaments (C) that terminate in a epithallial cells (white arrow) (L 3986120). Scale bar = 50 μm. (37) single layer of epithallial cells (black arrowhead) (UWC 15/59). Scale Vertical section through a mature, raised spermatangial conceptacle bar = 50 μm. (33) Vertical section of the inner thallus showing cell showing the mucilage plug (white arrowhead) that occludes the pore fusions (f) between adjacent medullary filaments (UWC 15/56). Scale opening and simple, spermatangial systems (black arrowheads) bar = 20 μm. (34) Vertical section of the outer thallus showing a confined to the conceptacle floor (UWC 15/56). Scale bar = 50 μm. (38) single layer of epithallial cells (e) subtended by a layer of Magnified view through a mature spermatangial conceptacle showing subepithallial initials (i). Note the cell fusions (f) between adjacent the pore canal lined with terminal, elongate initials (black arrowheads) cortical filaments (UWC 15/59). Scale bar = 20 μm. (35) Vertical that project into the pore canal as papillae (UWC 15/56). Scale section of the outer thallus showing a single layer of epithallial cells bar = 20 μm. (39) Magnified view of a mature carpogonial conceptacle (e) subtended by a layer of subepithallial initials (i). Note a cluster of with carpogonial branches comprising a single support cell (s), a bottle-shaped trichocytes (t) each separated by vegetative filaments hypogynous cell (h) with sterile cell (black arrowhead), and a (UWC 15/59). Scale bar = 20 μm. carpogonium (c) extended into a trichogyne (black arrows) that may project out of the pore. Note that the pore canal is lined with terminal, elongate initials (white arrows) that project into the pore canal as perpendicular to the conceptacle roof surface (Figure 39). papillae (L 3986120). Scale bar = 20 μm. (40) Vertical section through a fl Carpogonial branches developed across the oor of the mature carposporangial conceptacle showing a discontinuous central conceptacle chamber, comprising a single support cell, a fusion cell (black arrowhead) with peripherally arranged gonimoblast hypogynous cell and a carpogonium that extended into a filaments each terminating in a carposporangium (C) (L 3986120). trichogyne (Figure 39). Sterile cells were occasionally Scale bar = 50 μm. (41) Magnified view of the floor of a carposporangial conceptacle showing a discontinuous central fusion cell (black present on hypogynous cells (Figure 39). arrowheads) bearing a peripherally arranged gonimoblast filament After presumed karyogamy, carposporophytes (1–6) terminating in a carposporangium (C). The remains of unfertilised developed within female conceptacles and formed car- carpogonial branches (black arrow) persist across the dorsal surface of posporangial conceptacles (Figure 40). Carposporangial the central fusion cell (L 3986120). Scale bar = 20 μm. C.A. Puckree-Padua et al.: South African species of Chamberlainium 33

Figures 42–46: Chamberlainium occidentale tetrasporangial anatomy. (42) Vertical section through the outer thallus showing an early stage tetrasporangial conceptacle primordium with peripheral roof development (black arrowheads) and a protective layer of epithallial cells (e) (L 3986120). Scale bar = 50 μm. (43) Vertical section through the outer thallus showing a later stage tetrasporangial conceptacle primordium with peripheral roof development (black arrowheads) and tetrasporangial (t) initials arranged peripherally around a central columella (c). Note the persisting layer of protective epithallial cells (e) (UWC 93/220). Scale bar = 50 μm. (44) Vertical section through the outer thallus showing a maturing tetrasporangial conceptacle with peripheral roof development (black arrowheads) and tetrasporangial (t) initials, peripherally arranged around a central columella (c). Note the layer of protective epithallial cells (e) (UWC 93/220). Scale bar = 50 μm. (45) Vertical section through a mature, raised tetrasporangial conceptacle showing tetrasporangia (t) with stalk cells (white arrowheads), peripherally arranged around a well-defined central columella (C). Note, the sunken and unoccluded pore opening (white arrow) (UWC 93/220). Scale bar = 100 μm. (46) Magnified view of the pore canal of a mature tetrasporangial conceptacle showing terminal, elongate initials (black arrows) that project into the pore canal as papillae and the base of the pore canal sunken (black arrowheads) into the chamber with terminal, elongate initials near the base pointing downward. Note, the sunken and unoccluded pore opening (white arrow) (UWC 93/220). Scale bar = 20 μm. conceptacles were comparatively large, low-domed, and periphery of the conceptacle chamber (Figures 40, 41). raised above the surrounding thallus surface. Concep- The remains of unfertilized carpogonia persisted across taclechambersweretransverselyellipticalwithflattened the dorsal surface of the fusion cell (Figures 40, 41). Se- bottoms presumably caused by the growth of the goni- nescent carposporangial conceptacles appeared to be moblast filaments. Pore canals tapered towards the sur- shed as no buried conceptacles were observed. face, were lined by enlarged papillate cells that projected Tetrasporangial thalli are morphologically similar to into the pore canal and were orientated more or less gametangial thalli. Conceptacles were uniporate, low parallel to the conceptacle roof surface (Figure 40). In domed and raised above the surrounding thallus surface mature conceptacles, the cells lining the pore canal were (Figures 42–45). Conceptacle chambers were transversely more elongate than their inward derivatives. The base of elliptical to bean-shaped. The roof was nearly twice as the pore canal was sunken into the chamber and terminal thick along the pore canal and was 5–7 cells (including an initials near the base characteristically pointed down- epithallial cell) thick. The pore canal tapered towards the ward (Figure 40). A discontinuous central fusion cell was surface an was arched (Figure 46). The pore canal was lined present and 5–7 celled gonimoblast filaments (including a by elongated papillate cells that projected into the pore terminal carposporangium) developed along the canal and were orientated more or less parallel or nearly 34 C.A. Puckree-Padua et al.: South African species of Chamberlainium

perpendicular to the conceptacle roof surface (Figure 46). chambers (<300 µm for Chamberlainium and >300 µm for The roof was formed from filaments peripheral to the fertile Spongites); and 2) number of cell layers composing roofs of area and terminal initials were more elongate than their tetra/bisporangial conceptacles (≤8 cell layers for Cham- inward derivatives (Figures 42–44). Throughout the early berlainium and >8 for Spongites). Neither of these charac- development a protective layer of epithallial cells sur- ters is now useful to segregate these genera (Table 3). rounded the conceptacle primordium (Figures 42–44). This Chamberlainium impar (Puckree-Padua et al. 2020b) and protective layer was shed once the pore canal was near C. occidentale (this study) both have conceptacle chamber fully developed. The pore opening was unoccluded and diameters >300 um (up to 365 µm). Additionally, C. glebo- became slightly sunken below the surrounding roof sur- sum has up to nine cell layers comprising tetrasporangial face (Figures 45, 46). Throughout development of the conceptacle roofs. The two genera recognized within immature tetrasporangial conceptacle a prominent colu- Chamberlainoideae, Chamberlainium and Pneophyllum are mella of sterile filaments formed at the center of the still segregated by their different types of tetrasporangial conceptacle chamber (Figures 43, 44), which persisted to conceptacle roof development, type 1 or SUR-type (from maturity (Figure 45). The base of the pore canal was sunken filaments surrounding the fertile area) in the former versus into the chamber and terminal initials near the base point type 2 or COL-type (from filaments surrounding and within downward (Figure 46). Mature conceptacle floors were the fertile area) in the latter (see also Johansen 1976; sunken 14–19 cells (including the epithallial cell) below the Townsend 1981). Based on a combination of DNA se- surrounding thallus surface. Zonately divided tetraspor- quences and tetrasporangial conceptacle roof develop- angia at maturity were arranged at the extreme periphery of ment, we were able to correctly assign South African the conceptacle chamber and were attached via a stalk cell species previously placed in Spongites in either Chamber- (Figure 45). Senescent tetrasporangial conceptacles lainium (Puckree-Padua et al. 2020b, this study) or Pneo- appeared to be shed as no buried conceptacles were phyllum (Puckree-Padua et al. 2020a). The generic observed. Data on reproductive characters summarized in assignment of New Zealand species previously placed in Table 2. S. yendoi by Broom et al. (2008), which represent multiple Distribution: Confirmed by DNA sequences to be distinct species (Sissini et al. 2014), as well as topotype widely distributed (±1,200 km distance) along the west and material of S. yendoi from Japan, needs to be reassessed. southern west coasts, occurring from Lüderitz (Namibia) to For Chamberlainoideae Caragnano et al. (2018) re- Holbaaipunt (Western Cape Province), South Africa. The ported trichocytes to be absent or present; when present, species is disjunct in the southern part of its range of occurring singly or paired, but always terminal and never ±240 km. intercalary in the perithallus (cortex). However, our research has shown that for C. cochleare, C. natalense (Puckree-Padua et al. 2020b), and C. occidentale (this 4 Discussion study) trichocytes can occasionally occur in horizontal clusters of up to six cells, but each trichocyte is separated 4.1 Subfamily and generic assignment by one or more vegetative filaments (Table 3). Although this arrangement differs from that characteristic of Poroli- Puckree-Padua et al. (2020b) noted that there were no thoideae, where trichocytes are tightly packed in horizon- diagnostic characters that segregated the Corallinales tal fields without any vegetative filaments between them subfamilies Chamberlainoideae, Corallinoideae or Neo- (Kato et al. 2011), it is inconsistent with the description of goniolithoideae, and they also argued that it was prema- Chamberlainoideae proposed by Caragnano et al. (2018). ture to raise these subfamilies to the rank of family. Thus, Additionally, Caragnano et al. (2018) proposed that tri- taxa are assigned to these subfamilies based on DNA se- chocyte development in Chamberlainium was of the Jania quences of the commonly used markers for coralline sys- rubens type (Cabioch 1971; Johansen 1981). Observations tematics, nuclear encoded SSU or LSU and/or plastid of C. decipiens (van der Merwe et al. 2015), C. cochleare, encoded psbAorrbcL. Herein we used psbA and rbcL se- C. impar, C. natalense (Puckree-Padua et al. 2020b), quences to place three South African species in Chamber- C. capense and C. occidentale, suggest that trichocyte lainoideae, all of which were passing earlier under the development in Chamberlainium is rather of the Fosliella name S. yendoi. Caragnano et al. (2018) proposed two type. Here the overlying epithallial cell is shed prior to the morpho-anatomical characters to segregate Spongities trichome elongation and final development (Johansen (Neogoniolithoideae) from Chamberlainium (Chamberlia- 1981: 34, fig. 18); in the J. rubens type of development the noideae): 1) diameter of tetra/bisporangial conceptacle epithallial cell is not shed and instead the trichome C.A. Puckree-Padua et al.: South African species of Chamberlainium 35

Table : Morpho-anatomical characters considered by Caragnano et al. () to be informative in distinguishing among Chamberlainium, Pneophyllum and Spongites and the corresponding characters subsequently established for these taxa in South Africa.

Character Chamberlainium Chamberlainium Pneophyllum Pneophyllum Spongites (Caragnano et al. (Puckree-Padau et al. (Caragnano et al. (Puckree-Padau (Caragnano et al. ) b, this study) ) et al. a) )

Thallus thickness < mm ≤ mm Generally < mm (primary Up to several mm < μm, or fila- thalli), ≤ mm ments up  cells (secondary thalli) Thallus construction Dimerous or mono- Dimerous or monomerous, Dimerous Primarily dimerous, Dimerous or merous, but not both but not both becoming secondarily monomerous monomerous (can be both) No. of epithallial cells –– () –– Trichocyte arrangement Solitary, paired Solitary, paired, clustered in Solitary, paired None observed Solitary, horizon- (when present) (never in horizontal horizontal fields but sepa- tal fields, vertical rows) rated by vegetative rows filaments Development of tetra/ Type  Type  Type  Type  Type  bisporangial conceptacle roof Diameter of tetra/bis- < μmUpto μm< μm< μm> μm porangial conceptacle chambers No. of tetra/bispor- ≤ ≤ ()< Up to  > angial conceptacle roof cells Central columella pre- Absent or present Present, but may disinte- Absent or present Present, but may disin- Present, poorly sent/absent in tetra/ grate with tegrate with maturity developed bisporangial concep- maturity tacle chambers Distribution of tetra/ Peripheral or across Peripheral, but may appear Peripheral Peripheral Peripheral bisporangial in tetra/ chamber floor across the chamber floor as bisporangial concep- central columella tacle chambers disintegrates develops to project through the epithallial layer (Judson the same analyses of psbA sequences moderately supports and Pueschel 2002). These contradictory examples suggest C. capense sister to C. glebosum. The overall distributions of that the characterization of Chamberlainium (and possibly these three South African nearly endemic species overlap – also Pneophyllum, see Puckree-Padua et al. 2020a) will C. occidentale extends north into southern Namibia need emending. We propose that any emendation should (Figure 1) – but each has unique ranges (discussed below). avoid including characters that are either highly variable, The analyses of both genes also moderately (psbA) to or are difficult to interpret. strongly (rbcL) support another endemic South African species, C. cochleare, as sister to these three species. However, C. cochleare is distributed further east along the 4.2 Species relationships south coast and does not overlap the ranges of C. capense, C. glebosum and C. occidentale. The phylogenetic re- The interspecific sequence divergence values from the lationships of these four species with the three other psbA and rbcL genes indicate that C. capense, C. glebosum named South African Chamberlainium species, C. agul- and C. occidentale are distinct species (Tables S2, S3). This hense, C. impar and C. natalense and with the NE Pacific is supported by the ABDG analysis reported in Puckree- species C. tumidum and C. decipiens, are unresolved in the Padua et al. (2020b). These species occur in a fully sup- rbcL tree (Figure 3), whereas in the psbA tree, C. agulhense, ported clade in analyses of both the psbA and rbcL genes, C. impar and C. natalense are moderately to strongly sup- although the relationships among the species are unre- ported as sister to two unnamed Chamberlainium species. solved. Both Bayesian and ML analyses of rbcL sequences Analysis of additional markers, such as LSU, SSU or CO1 fully support C. capense sister to C. occidentale, whereas may be helpful in resolving this relationships. 36 C.A. Puckree-Padua et al.: South African species of Chamberlainium

4.3 Species accounts Aside from these collective field characters, histolog- ical examination in the laboratory has shown that thallus Collectively, the geographic distributions, substrate, construction, trichocyte arrangement, number of epi- habit, habitat, growth form and color of living thalli are thallial cell layers and the structure of the tetrasporangial useful characters for field identification of the various conceptacle pore canal are useful anatomical characters South African species of Chamberlainium thus far (Table 4) to distinguish some species. Chamberlai- described (Table 4). Of the named Chamberlainium spe- nium agulhense is the only dimerous species. While tri- cies, C. agulhense has the most restricted distribution, chocytes have not been observed in C. agulhense and occurring epilithically on the primary bedrock mostly in C. glebosum, they occurred singly to paired in C. capense the high intertidal along the South African warm and C. impar, and often occurred clustered in small hori- temperate south coast (Benguela-Agulhas Transition zontal fields in C. cochleare, C. natalense and C. occi- Zone; Smit et al. 2017) within a narrow 10 km range (van dentale. Most species have only a single layer of epithallial der Merwe et al. 2015). Chamberlainium cochleare and cells, but C. agulhense can have up to 3 layers of epithallial C. natalense have wide distributional ranges, but are cells; C. impar is always multi-layered with up to 9 layers of restricted to the warm temperate south coast (Benguela- epithallial cells. The tetrasporangial conceptacle pore is Agulhas Transition Zone and the Agulhas Marine Prov- not occluded in C. agulhense, C. glebosum and C. occi- ince; Smit et al. 2017) and subtropical east coast (East dentale, although in the last species the pore opening is Coast Transition Zone; Smit et al. 2017) (Puckree-Padua slightly sunken below the surrounding roof. The projecting et al. 2020b). Chamberlainium cochleare is epilithic on the corona above the tetrasporangial conceptacle pore may be primary bedrock in the mid to low intertidal zone and is useful for distinguishing C. capense from all other South characteristically associated with the gardening limpet, African species of Chamberlainium except for C. natalense Scutellastra cochlear, whereas, C. natalense is found (Puckree-Padua et al. 2020b). As in C. natalense, the corona largely epilithic on boulders in intertidal rock pools. appears to develop from filaments near the upper half of Chamberlainium capense, C. impar, C. glebosum and the tetrasporangial conceptacle roof, directly adjacent to C. occidentale are all largely cold temperate species the pore canal. However, C. natalense is mostly found on restricted to the cool temperate west coast (Benguela boulders and pebbles in rock pools and has a thinner Marine Provice; Smit et al. 2017) (Puckree-Padua et al. thallus (up to 800 µm) that is entirely smooth (lacking 2020, this study). Along this coast C. capense (±43 km) protuberances). Moreover the two species do not have and C. impar (±170 km) have restricted southerly distri- overlapping distributions with C. capense found in cool butions and are epilithic in the mid to low intertidal zone. temperature waters whereas C. natalense is found in warm Chamberlainium glebosum has a wide, but disjunct dis- temperate to subtropical waters. In C. cochleare and tribution (±220 and ±200km)withagapnorthandsouth C. impar, the tetrasporangial conceptacle pore is occluded of Lambert’s Bay. The species is epilithic on the primary by a mucilage plug, but the two species are morphologi- bedrock throughout the intertidal zone. Similar to cally distinct (Table 4). Except for the corona of filaments C. cochleare and C. natalense, C. occidentale has a wide that occlude the tetrasporangial conceptacle pore in distribution (±1,200 km) along the African southern west C. capense, anatomically there are no useful, observable coast occurring on bedrock throughout the intertidal characters with a light microscope that separate this spe- zone and epizoic on the shells of live molluscs; the species from either C. glebosum or C. occidentale. However, of cies has a disjunction in the southern part of its range of all the South African Chamberlainium species thus far ±240 km. While gross morphology (growth form and described, C. glebosum is the most protuberant. thallus color) is a useful character for separating taxa across biogeographic boundaries (the warm temperate and subtropical species are generally encrusting to 4.4 Range-restricted coralline endemics smooth; the cool temperate species are generally warty to lumpy, becoming highly protuberant), it is not useful For most marine algae, range-restrictions seem to be within biogeographic regions. Chamberlainium impar, associated with biogeographic boundaries or transition however, has a unique yellowish to yellow-brown thallus zones (Anderson et al. 2009; Montecinos et al. 2012). While that is pachydermatous and often highly confluent. Of all the underlying mechanisms that result in coralline algal the species C. cochleare is the only one in which indi- range-restrictions remain unknown (Hind et al. 2016), vidual thalli are not discernible due to their fusing to biogeographic breaks offer a plausible explanation for form large expanses. some species. There are now several examples of coralline Table : Features found to be informative for distinguishing among the various South African species of Chamberlainium thus far described.

Character Chamberlainium Chamberlainium Chamberlainium coch- Chamberlainium Chamberlainium Chamberlainium Chamberlainium agulhense (van der capense (this study) leare (Puckree-Padua glebosum (this impar (Puckree- natalense (Puckree- occidentale (this study) Merwe et al. ) et al. b) study) Padua et al. b) Padua et al. b)

Endemic Yes Yes Yes Yes Yes Yes No (Namibia and South Africa) Geographical Restricted distribu- Restricted distribution Widely distributed Disjunct distribution Restricted distribu- Widely distributed Widely distributed distribution tion (± km) along (± km) along the (±, km) along the (± and ± km) tion (± km) along (±, km) along the (±, km) along the the southern west southern west coast southern and eastern along the west coast the southern west southern west and west and southern west coast coasts coast eastern coasts coasts Substrate Epilithic on bedrock Epilithic on bedrock Epilithic on bedrock; Epilithic on bedrock Epilithic on bedrock Largely epilithic on boul- Epilithic on bedrock; and boulders; epizoic epizoic on mollusc ders and pebbles; occa- epizoic on mollusc on mollusc shells shells, especially sionally epilithic on shells S. cochlear bedrock; epizoic on winkle shells Habitat High (mostly) to mid- Mid-intertidal (rock Mid- to low intertidal. High to mid-shore Mid- to low intertidal. Mid- to low intertidal rock High to low (largely) intertidal pools mostly) to low (mostly) to occasion- pools. intertidal

intertidal. ally on the low shore. of species African South al.: et Puckree-Padua C.A. Growth form Encrusting (smooth) Encrusting (smooth), Encrusting (smooth) Lumpy, becoming Encrusting (smooth), Encrusting (smooth) Encrusting (smooth) to becoming variably highly protuberant orbicular becoming warty (mostly) to lumpy (mostly) and conﬂuent lumpy, becoming slightly protuberant slightly protuberant Habit Individual thalli Individual thalli Individual thalli not Individual thalli Individual thalli Individual thalli discern- Individual thalli discernible, not discernible, not fusing discernible, fusing to discernible, not discernible, not ible, not fusing discernible, not fusing fusing form large expanses fusing fusing Color of living Brownish-pink Bright to dusky pink Greyish Brownish pink to gray Yellowish to yellow Blue-gray Brownish beige thalli (in well-lit brown conditions) Thallus Dimerous Monomerous Monomerous Monomerous Monomerous Monomerous Monomerous construction Trichocyte None observed Common, solitary Common, solitary None observed Rare, solitary to Rare, solitary (mostly) to Common, solitary arrangement (mostly) to paired (mostly) to clustered, paired clustered, up to  (sepa- (mostly) to clustered, up to  (separated by rated by vegetative up to  (separated by

vegetative filaments) filaments) vegetative filaments) Chamberlainium No. of epithallial – (mostly –) – ()  cell layers Mature tetraspor- Pore opening Pore opening occluded Pore opening occluded Pore opening Pore opening Pore opening occluded Pore opening unoc- angial pore unoccluded by a corona of by a mucilage plug unoccluded occluded by a muci- by a corona of filaments cluded; pore slightly characteristics filaments lage plug sunken below the surrounding roof 37 38 C.A. Puckree-Padua et al.: South African species of Chamberlainium algae with restricted geographical ranges (e.g., Hind et al. 5 Conclusions 2016; Pardo et al. 2014; Peña et al. 2014, 2015; Wilson et al. 2004), and South Africa is no exception, being bathed and DNA sequencing is profoundly changing our understand- fl in uenced by three oceans, the western Atlantic (with its ing of the composition of the South African non-geniculate cold Benguela Current), the eastern Indian (with its warm coralline flora. Thus far, none of the species previously Agulhas Current), and the Southern Ocean to the south reported from South Africa based on morpho-anatomy, (with its cold Antarctic Circumpolar Current). Due to their and whose type localities are on other continents, is proximity, the former two ocean currents have the greatest correctly applied in South Africa, e.g., P. discoideum (as influence on the South African marine biota. The effects of S. discoideus) from South America (Puckree-Padua et al. these ocean currents have resulted in two marine provinces 2020a), S. yendoi from Japan (Puckree-Padua et al. 2020b), and two biogeographic transition zones (Figure 1; see Smit and Phymatolithon repandum from Australia (Maneveldt et al. 2017). The western biogeographic transition zone et al. 2020). Very likely none of the remaining 19 species, (Benguela-Agulhas Transition Zone) is located within the with their type localities on other continents, are present in Cape Peninsula and experiences rapid changes in tem- South Africa (see Maneveldt et al. 2016 for the most recent perature (Bolton and Anderson 1997; Leliaert et al. 2000; list). In addition, seven distinct species were passing un- Smit et al. 2017). This biogeographic break seems to be der S. yendoi in South Africa, none of them are that species, largely responsible for the distributional ranges we and all are classified in the recently erected genus, observed for the South African species ascribed to Cham- Chamberlainium. We have just scratched the surface berlainium (Puckree-Padua et al. 2020b; van der Merwe sequencing DNA from only two genera, Spongites and et al. 2015, this study) and Pneophyllum (Puckree-Padua Phymatolithon, of the 18 reported from South Africa et al. 2020a). Within Chamberlainium, at least three species, (Maneveldt et al. 2016). We anticipate, with further C. agulhense (van der Merwe et al. 2015), C. impar (Puckree- research, finding many additional misapplied names and Padua et al. 2020b), and C. capense (this study) have undescribed taxa. restricted geographic ranges, all of them associated with biogeographic boundaries or transition zones. Chamber- Acknowledgments: We thank Wilson Freshwater, DNA lainium agulhense has the most restricted distribution Analysis Core Facility, University of North Carolina, Wil- (10 km range), occurring along the South African warm mington for providing sequencing support and Todd temperate south coast in the western biogeographic tran- Vision for providing research space and equipment to sition zone (Benguela-Agulhas Transition Zone; Smit et al. PWG. We are grateful to Arley Muth for collecting material 2017) (van der Merwe et al. 2015). The precise reason for this in Chile and to the Amsler lab (University of Alabama, very narrow range is unclear, but another non-geniculate Birmingham) for collecting material in Antarctica. coralline alga, Heydrichia cerasina Maneveldt et E.van der Author contributions: All the authors have accepted Merwe has the same biogeographic distribution (Man- responsibility for the entire content of this submitted eveldt and van der Merwe 2012), suggesting that the Cape manuscript and approved submission. Agulhas region, where these species occur, represents a Research funding: We thank the Department of region of biogeographic interest. Chamberlainium capense Biodiversity and Conservation Biology at the University (±43 km) and C. impar (±170 km) are both cool temperate of the Western Cape for providing funding and research species and are restricted to the southern regions of the equipment. The South African National Research Benguela Marine Province. The two species have a small Foundation (NRF), the South African National Botanical overlap (±4 km) in their northern and southern distribu- Institute (SANBI) through its Foundational Biodiversity tions respectively. Interestingly, C. occidentale, a cool Information (FBIP) and SEAKEYS programmes, and the temperate species with a wide distribution (±1,200 km) Department of Science and Technology (DST) through its along the African southern west coast, also has a disjunc- African Coelacanth Ecosystem (ACEP) and Phuhlisa tion in the southern part of its range of ±240 km. Cham- programmes, are thanked for research funding, bursaries berlainium glebosum could also be considered a range- and travel awards to GWM and CAP-P. PWG acknowledges restricted endemic because within its disjunct distribution a private family trust for research support. (±220 and ±200), the species appears to be restricted, with a Conflict of interest statement: The authors declare no gap north and south of Lambert’s Bay. conflicts of interest regarding this article. C.A. Puckree-Padua et al.: South African species of Chamberlainium 39

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Peña, V., Rousseau, F., De Reviers, B., and Le Gall, L. (2014). First Bionotes assessment of the diversity of coralline species forming maërl and rhodoliths in Guadeloupe, Caribbean using an integrative Courtney A. Puckree-Padua systematic approach. Phytotaxa 190: 190–215. Department of Biodiversity and Conservation Peña, V., De Clerck, O., Afonso-Carrillo, J., Ballesteros, E., Bárbara, I., Biology, University of the Western Cape, P. Barreiro, R., and Le Gall, L. (2015). An integrative systematic Bag X17, Bellville 7535, South Africa approach to species diversity and distribution in the genus https://orcid.org/0000-0001-7684-0610 Mesophyllum (Corallinales, Rhodophyta) in Atlantic and Mediterranean Europe. Eur. J. Phycol. 50: 1–15. Puckree-Padua, C.A., Gabrielson, P.W., Hughey, J.R., and Maneveldt, G.W. (2020a) (in press). DNA sequencing of type material reveals Pneophyllum marlothii comb. nov. from South Courtney A. Puckree-Padua is currently employed as a Doctor of Africa and P. discoideum comb. nov. (Chamberlainoideae, Marine Biology at the Cape Peninsula University of Technology, South Corallinales, Rhodophyta) from Argentina. J. Phycol. Africa. She was awarded a PhD in Algal Taxonomy by the University of https://doi.org/10.1111/jpy.13047‐20‐081. the Western Cape, where her postgraduate research was conducted. Puckree-Padua, C.A., Haywood, A., Gabrielson, P.W., and Courtney used an integrated taxonomic approach to resolve the Maneveldt, G.W. (2020b) (in press). Reassignment of some South biodiversity of species previously assigned to the genus Spongites in African species to Chamberlianium, with a comment about the South Africa. Her more recent research includes resolving the recognition of families of Corallinales (Rhodophyta). Phycologia phylogenies within the Corallinales from Chile and Antarctica. 59, https://doi.org/10.1080/00318884.2020.1975797. Rösler, A., Perfectti, F., Peña, V., and Braga, J.C. (2016). Phylogenetic Paul W. Gabrielson relationships of Corallinaceae (Corallinales, Rhodophyta): Biology Department and Herbarium, taxonomic implications for reef-building corallines. J. Phycol. 52: University of North Carolina at Chapel Hill, 412–431. Coker Hall CB 3280, Chapel Hill, NC, 27599- Sissini, M.N., Oliveira, M.C., Gabrielson, P.W., Robinson, N.M., 3280, USA Okolodkov, Y.B., Riosmena-Rodríguez, R., and Horta, P.A. (2014). https://orcid.org/0000-0001-9416-1187 Mesophyllum erubescens (Corallinales, Rhodophyta): so many species in one epithet. Phytotaxa 190: 299–319. Smit, A.J., Bolton, J.J., and Anderson, R.J. (2017). Seaweeds in two Paul W. Gabrielson, a Herbarium Research Associate and Adjunct oceans: beta-diversity. Frontiers Mar. Sci. 4: 404. Professor of Biology at the University of North Carolina, Chapel Hill, Stamatakis, A. (2006). RAxML-VI-HPC: maximum likelihood-based does research on algal systematics. His two post-docs were with the phylogenetic analyses with thousands of taxa and mixed esteemed phycologists Dr. Gerald T. Kraft and the late Dr. Robert F. models. Bioinformatics 22: 2688–2690. Scagel. Paul began to re-tool from morpho-anatomy to DNA Stearn, W.T. (1973). Botanical Latin. David and Charles, Newton Abbot. sequencing while teaching at a small, liberal arts college for nine Stephenson, T.A. and Stephenson, A. (1972). Life between tidemarks years, before returning to North Carolina. Paul collaborates with on rocky shores. Freeman, San Francisco. phycologists worldwide sequencing field-collected and type Thiers, B.M. (2020). [Continuously updated electronic resource]. Index specimens of seaweeds from the 18th, 19th, and early 20th centuries, herbariorum: a global directory of public herbaria and associated especially coralline red algae. staff. New York Botanical Garden’s Virtual Herbarium, Available at: http://sweetgum.nybg.org/ih/. Gavin W. Maneveldt Townsend, R.A. (1981). Tetrasporangial conceptacle development as a Department of Biodiversity and Conservation taxonomic character in the Mastophoroideae and Biology, University of the Western Cape, P. – Lithophylloideae (Rhodophyta). Phycologia 20: 407 414. Bag X17, Bellville 7535, South Africa van der Merwe, E., Miklasz, K., Channing, A., Maneveldt, G.W., and [email protected] Gabrielson, P.W. (2015). DNA sequencing resolves species of https://orcid.org/0000-0002-5656-5348 Spongites (Corallinales, Rhodophyta) in the Northeast Paciﬁc and South Africa, including S. agulhensis sp. nov. Phycologia 54: 471–90. Gavin W. Maneveldt is a Professor of Marine Biology in a small Wilson, S., Blake, C., Berges, J.A., and Maggs, C.A. (2004). department at the University of the Western Cape, South Africa. He Environmental tolerances of free-living coralline algae (maërl): was initially interested in pursuing research into marine herbivore- implications for European marine conservation. Biol. Conserv. algal interactions, but the lack of suitable names for most of the 120: 279–289. coralline algae he encountered, quickly changed that focus. Since Woelkerling, W.J., Irvine, L.M., and Harvey, A.S. (1993). Growth-forms obtaining his PhD, Gavin’s interest shifted almost entirely to the in non-geniculate coralline red algae (Corallinales, Rhodophyta). taxonomy and systematics of the non-geniculate coralline red algae. Aust. Syst. Bot. 6: 277–293. Gavin collaborates extensively with phycologists of similar interest worldwide, lending his unique expertise in histology and morpho- Supplementary Material: The online version of this article offers anatomy to the understanding of the diversity of the non-geniculate supplementary material (https://doi.org/10.1515/bot-2020-0074). coralline red algae.